Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the non-catalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase, Plk, is a functional homologue of Saccharomyces cerevisiae Cdc5. Here we show that Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a cdc5-1 temperature-sensitive mutation, and to localize to these subcellular structures. Our data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization.